Method and apparatus for determining toxicity



Jan. 16, 1951 M. EQBENESH 2,538,498

METHOD AND APPARATUS FOR DETERMINING TOXICITY Filed Sept. 5, 1948 3 Sheets-Sheet l 10x10 uouw ALL GLASS CHAMBER L 4 SUCTION 42 (IAWATER) EXIT 45 SAMPLING TUBE CARRIAGE BURETTE STAINLESS STEEL TUBING 23 CYUNDER CARRIAGE BOX I8 20 GEAR BOX (ADJUSTABLE) 'I'cLurcH Z SE GEAR BOX [6 (FIXED) 7 AIR SY NCHRONOUS M OTOR 3 Sheets-Sheet 2 M. E. BENESH METHOD AND APPARATUS FOR DETERMINING TOXICITY Jan. 16, 1951 Filed Sept. 3, 1948 MATTHEW E. BENESH Jan. 16, 1951 M. E. BENESH METHOD AND APPARATUS FOR DETERMINING TOXICITY 3 Sheets-Sheet 5 Filed Sept. 3, 1948 6 .m M v m a H s E N E B E W E H T T A M 1w mm mm 9 6 mm mm vw r 4 mm U mm 9 mm 0 on ow .w mm

mm hm Patented Jan. 16, 1 951 METHOD AND APPARATUS FOR DETEBWING TOXICITY Matthew E ward Benesh, Cicero, in.

Application September 3, 1948, Serial No. 50,761

4 Claims.

The present invention relates to testing apparatus and more particularly to a gassing chamher for rapidly determining the toxicity of various fluids. An important object of the present invention is to provide an improved apparatus for the rapid determination of the efiect of toxic compounds on animal life. Another object is to pro vide such apparatus that is capable of testing a greatly increasednumber of difierent compounds in a given period of time. A further object is to provide means for the rapid and accurate testing of substances of low volatility. Other equally important objects will more plainly appear from the detailed drawings and specification presented in exemplification but not in limitation of the present invention. Like reference characters represent like parts in the drawings which illustrate diagrammatically in:

. Figure 1, a diagram illustrating a preferred ar-v rangement of the various cooperating elements of the apparatus.

Figure 2, a diagram illustrating details of the piston and cylinder and showing a rear view of the burette assembly shown in Figure 1.

Figure 3, a View in perspective of a detail of the carriage assembly.

Prior gassing chambers possessed certain inherent disadvantages among whichare the following six serious defects. First, the time .consumed. Prior chambers require a period of equilibration to establish the desired concentration before animals can be exposed in it. The equilibration period varies with the rate of air flow through the chamber and can be described bythe equation:

T 4.6 chamber volume 99* rate of air flow in which T99 equals the time required to establish 99 of the desired equilibrium concentration. In the case of the 40:0 liter chamber operating with an air flow of 250 liters per minute, 7.5 minutes are required for equilibration. For example, a ten minute exposure requires an experimental period of approximately 18 minutes.

Second,- the prior art hazards. When equilibrium has been established, the animals are insorted into the chamber, and after the desired pcriod of exposure they are withdrawn. During requires the use of a mask by the operators, and in testing powerful sensory irritants unmasked laboratory personnel not in the immediate vicinity of the chamber may be harmed.

Third, the excessive amounts of material required. Since 250 liters of air per. minute for approximately 20 minutespass through prior chambers during the determination of a ten minutes exposure mortality figure, it will be apparent that about 5,000 times the desired concentration in milligram per liter'of toxic agent is required for a single determination. often submitted in 10 or 20 gram quantities, it will be apparent that a single determination often exhausts the sample.

Fourth, the adverse dilution factor. In prior gassing chambers the toxic agent and the diluting air are introduced and may be varied separately. Since the usual rate of air flow through the chamber is'250 liters a minute, the toxic agent must be introduced into the chamber at a concentration 250 times that of the desired final concentration. In order to achieve this high concentration it is often necessary to heat the bubbler which contains the toxic agent to a point where the volatility is at least 50 times that of the desired final concentrationwithin the chamber. This procedure often results in the partial decomposition of the toxic agent before it can be tested. g r 1 Fifth, the admission of impurities. Since prior gassing procedures depend upon the vaporizationof the toxic material by forcing nitrogen or air through a bubbler containing the material maintained at a constant temperature, it will be apparent that impurities of higher or lower vapor pressure present in the material to be tested will seriously affect the accuracy of the results obtained. In addition prior gassing cham-- bers provide'insufiicient means to determine-the vapor pressure and volatility of the compound necessary to secure the. desired concentration of the agent in the chamber. Finally, it will be apparent that the introduction of mixtures of two or more materials into prior chambers simultaneously is a cumbersome and unreliable procedure.

Sixth, the corrosion and deposition inescapable in the prior apparatus. The rate of air flow through prior art chambers is determined by means of an orifice meter. Corrosion of the orifice plate, or deposition of materials upon it tend to introduce serious errors in measurement of the rate of air flow through the chamber.

' In the present-invention a toxic liquid may be Since newagents are displaced into an atomizer at any required rate, for example from 0.015 cc. to 21.4 cc. per minute. The rate of displacement depends upon the rate at which a piston is forced into a cylinder containing mercury. This piston speed is controlled by means of a change gear box. The atomized liquid is vaporized from a large surface by means of a warm air stream, and is then diluted with a carefully metered air stream before passing over the test animals. Because of its automatic nature the present apparatus permits testing of compounds at a speed hitherto unattainable. It has been found to be accurate, convenient, devoid of hazards, and well adapted to thestudy'of mixtures of toxic compounds.

The principle of the apparatus it of the present invention is illustrated in the diagram in Fig- 1. For example, an 18 liter glass chamber into which the animals are inserted is shown at" 42. Chamber 42 rests upon a glass plate 48 which serves as a floor. lhe chamber 42' can be raised from the glass floor 48' by appropriate hand lever operated rack and pinion means not shown. to permit insertion and removal of the animals to be exposed. A gastight seal is maintained by means of rubber gaskets 49. The chamber 42 is constructed with double walls shown at 41, and a suction of preferably 1 inches of water is maintained at 45 between the walls 4 to prevent leakage of toxic material.

A constant and accurately metered rate of air flow, for example 180 liters per minute, is maintained through the chamber 42 by means of a metered air pump Hi. This pump is operated through shaft M by positive gear box l2 from: a synchronous motor ll through connection i3. Motor H is also engaged through clutch I l to gear box I8, shaft 59 to gear box 20, all of which control the delivery of the toxic fluid so that even if the motor II is not constant, the proportion of toxic agent to the total air remains constant.

The toxic liquid is floated on a column of mercury 24 maintained in the burette assembly 26. One end of burette assemblyZG is connected to an atomizer 29 and on the other to a steel cylinder 23 containing mercury 24 as shownin Fig. 2. A stainless steel piston 22, preferably /3 or inch in diameter, fits into cylinder 23 and is tightly sealed with neoprene gaskets 66 so that no mercury leak can occur.

The piston 22 is attached to carriage assembly 2! and connected through gear box 20 to a com.- pound set of adjustable gears f8 driven by motor H which also drives the metered air pump I The rate at which the piston 22 enters the cylinder 23 and causes mercury 24 .torise and displace the toxic liquid l'fill into the atomizer 29 may. be controlled by the adjustable. gear box L8 in such a manner that more than 350 different: ratesare possible. The gears. at. lzflmay be adjusted so that the piston 22 may be provided with. a. rate. of travel ranging from 0.075 to 8.34. inches. per minute. This range can, for. example, be. divided by proper. adjustment of the gears at 18; into. 385 separate steps. This makes it. possible to select a gear setting such that the ratev of, delivery is usually the exact amount desired, and is always within 2% of the amount required. The threaded shaft 52 upon which the carriage 2|v moves is preferably machined with 24 threads to the inch. Any appropriate table canbe computed bywellknown methods for providing the desired. relationship of the gear settings at I8 to the distance which the piston 22 will be moved by the shaft 52 in a given time period. In. order to displace a given volume of material in a given time it is necessary only to refer to such a table to determine the proper gear settings in the table and adjust accordingly.

The main air stream at 35 is arranged so that 158 liters per minute go directly into the chamber 42, while 22 liters per minute enter a simple compressor indicated at 33 where it is compressed to provide a pressure ranging from 50 to 70 pounds. The compressed air may be heated by a suitable air heater at 3!, used to atomize the liquid which has been forced into the atomizer assembly 2-9. The resulting spray is deposited in a fine film upon the interior wall of spiral condenser 36, which may be heated with steam or hot water introduced at 31, and is vaporized from the wall by means of the same air which has performed the atomization. The air containing toxio'vapor IE0 is then returned to the main air stream and passes through opening 4! over the animals in the glass chamber 42-, leaving. the chamber 42' through openings 43 and exit 45.

Illustrated in, the lower portion of Fig. 2 is a front view of piston 22 and cylinder 23. The cylinder 23 is provided at its rearward end with a flangev t3 and a compression plate 9% held to the flange 9135 by bolts. 65 or other suitable attaching means. Neoprene gaskets 68 are: held between steel collars E l around the. piston 22. and within the cylinder. 23 and arev sealed: in compression by tightening. the plate 9.4.

The forward end of the cylinder 23' is provided with a flanged elbow member 68. to which is affixed the sealing clamp 69' by means of bolts I0 or other appropriate means. Lead gasket. 1 l. and shellac seal 12 also helps to prevent leakage around the tube 2?. The tube 2! is preferably 20-gauge stainless steel tubing and leads to the burette assembly 26 as shown in Fig. 1 and Fig. 2.

The burette assembly previously indicated by a front view at 26 in the overall diagram shown in Fig. l, isshown in the upper portion of Fig. 2 by a rear view thereof. Assembly 25 is connected to the cylinder 23 by means'ofthe stainless steel tubing 21 which is provided with the safety spring valve shown at T3 and with the 2-way stop cock l4- controlling the outlet 23 to a mercury reservoir. The 3-way stop cock shown at 15'may be preferably arranged to be electrically actuated by well-known solenoid means. Stop cock H which controls the releaseof mercury through the outlet 82 communicates with cock it. Positioned above the outlet valve H is a graduated capillary member 85 leading to a well-known mercurywater interface arrangement in cylinder 18 which contains the mercury 24 and the contacting Water 19.

As shown in the upper half of Fig. 2, the first mercury-water interface cylinder 18 communicates with the stop cock 8% which is used for evacuating the burette assembly 26. when it is being filled with mercury 22 and water 19. In this operation vacuum is applied through the stop cook 88 to remove all traces of compressible gas. so that the entire system. from the cylinder 23 to. the atomizer assembly 22 contains noncompressible liquid.

The stop cock 82'} is also connected with a second mercury-wa.ter interface cylinder 33- containing water at it and mercury at 2%. The lower portion of cylinder 8-3 in communication through tube 32' with the stop cook it and the stop cook 84. When stop cock at is turned 99 to the right the mercury 24. is withdrawn from the graduated cylinders $5,.- and- 85 through the 5, outlet81 which permits the toxic agent to be drawn into the burette assembly '26 through the conduit 28 from the atomizer assembly 29. The stop cock 85 is connected to the cylinders 85 and 88 by means of conduit 83. Cylinders 85 and 86 are provided with suitable graduations for measuring theamount of mercury to be displaced. This arrangement permits the toxic liquid I80 to be floated on the mercury 24 in cylinder-86 and its volume measured by displacement of the mercury as shown on the graduated scale on cylinders 85 and 8.6.

The 3-way stop cock i5 is shown in an operating position for a routine experiment. In this position mercury flows through thegraduated capillary tube BI and cylinders 86, 85, 83 and i8 displacing toxic agent I88 through the outlet tube 28 to the atomizer assembly 29. Before the starting contact for motor is engaged, the by-pass 38 shown in Fig. 1 is opened and the stop cock 15 is turned 90 to the left. The mercury 24 then 'flows horizontally from stop cock 15 through conduit 89 and through open stop cock 84 to cylinders 85 and 85-. In this way the relative positions of the mercury 2L 'and the water 19 in cylinders 18 and 83 are not disturbed until the starting contact for motor H is engaged. At'the start of the test or experiment the mercury 2d is set at a given mark in capillary 8| and is returned to this mark at the end of the experiment by turning stop cock 84 to a neutral position, next opening stop cock E5 to the mercury reservoir 82 and withdrawing mercury by means of stop cock Tl until the original mark is reached on capillary 8|. In Fig. 3 the carriage assembly 2| includes the carriage gear box 28 arranged in operative relationship with the threaded shaft 52 for advancing the piston 22, and with the adjustable gear box I8 shown in Fig. l. The threadedshaft 52 is preferably provided with 24 threads per inch. I

The accuracy of the machining can readily be tested by causing the piston 22 to be moved into the cylinder 23 for a given number of screw threads. This causes the-mercury 24 torise in the burette assembly 26. A mercury water interface is provided at 78 and at 83 in the burette 25 so that at the start the mercury 24 may be set at a given mark in graduated capillary 8%. After the piston 22 has been moved forward,-the mercury 2G can be withdrawn until it reaches the same given mark, and can then'be weighed. It has never been found to differ more than 0.3% from'thefdisplacement calculated from the cross sectional area of the piston'22 and the distance it moves as measured by a suitable revolution counter 5'l placed on the shaft 52 as shown in Fig. 3. I

A table is available which relates the gear settings to the distance which the piston will move on the screw in a given time period. In order to displace a given volume of material in a given time it is necessary'only to look up the proper gear settings in the table and adjust accordingly. I 1

- Referring again to Fig. 3, suitable clutch means 53 and 54 may be employed for releasing starting contact member 58 and stopping contact member 5| so that they may be set at any chosen distance apart on their respective shafts 6B and 5f. Counter 55 is used for determining the setting 'of starting contact 58, and counter 56 is likewise used for stopping contact 5|. Each number on counters 55 and 56 represents one-full turn of the threaded shaft 52. The numerical difference between the reading on counter 55 and that on counter 56 represents the distance expressed in screw threads separating the starting and stopping contacts 58 and 5|. Counter 51 counts the number of complete turns made by threaded shaft 52 and Vernier counter 58 counts fractions of a turn made by shaft 52. At the beginning of the experiment the reading is'noted on counters 51 and 58 and at the end of the experiment the reading onthese counters 51 and 58 is again noted. The numerical difference between these two readings represents the exact number of threads (on-shaft 52) which piston 22 has moved forward during the experiment. From this distance'the exact volume of toxic fluid I80 displaced by mercury 24 through tube 28 may be readily computed by means of the varioustables referred to herein.

Control mechanism Y Fig. 2 illustrates in perspective a skeleton view of a front elevation of a part of the carriage as- 'sembly 2|. (The carriage 21 as diagrammed in Fig. lshows a top view representation of members 58 and 5| to clarify that part of Fig. 1.) It

r includes the end support members 9| and 92 and longitudinal support members 95. The main threaded shaft 52 is driven through change gear box 28 by means of a system ofgears which rotatesia nut that is in engagement with shaft '52 and thereby causes shaft 52 and carriage 2| to travel longitudinally in a forward and rearward direction in a manner'well-known in the mechanical arts." The piston 22 preferably has a suitable floating bearing connection with end support 92 so that maximum accuracy may be assured by causing the piston 22 to beindependently rotated on its longitudinal axis during its longitudinal advance against the column of mercury2'4 in the cylinder 23." This is accomplished by means of the pinion attached to piston 22 and engaging driving gear 63. Drive gear 63 is rotated by the driving shaft 59 from the in dependent driving motor 62. Shaft 52 is providedwith a suitable revolution counter 51 ccoperating with a Vernier counter 58 for indicating" fractions of a revolution, so that the exact distance of'travel of piston 22 may be accurately indicated. f

[Suitably mounted in the end supports 9| and 92 are the threaded shaft 60 used for starting con- ,trol, and the threaded shaft 6| used for stop:

ping -control. identical number of threads per inch, preferably 24,. that are provided in shaft 52. vThreaded shafts 68 and 6| are provided withrevolution counters 55 and 56; respectively, for setting members 50 and 5|on shafts 80 and 6| at their chosen positions. Trip members 5i and 50 are in threadedengagement with the threaded shafts 6| and BI], respectively. Members 58 and 5| travel with carriage 2|. When member 58 reaches the end of its travel, it is arranged to actuate a starting solenoid switch lever and in a like manner trip member 5| when it reaches the end of its concurrent travel with carriage 2|, it is arranged to actuate a stopping solenoid switch lever. These ponents of the apparatus of the present invention in the following manner.

' The 3-way valve at 15 is actuated by a servo- Both of these shafts have the I motor; the, clutch. at I! is actuatedby a. servo-- motor; and the by-passvalve at 38 is also servomotor operated. The compressor switch at 33, the meter switch at l5 and the servo-motor switches are arranged to be. energized after contact members 50 and 5 l are set at the desired positions- Each of these members is arranged to. actuate a well-known micro-switch which in turn concurrently actuates clutch l1, and 3-way valve 15, in a manner well-known to those skilled in the art of servo-motors.

Referring to Fig. 2; before beginning the operation it is necessary to turn the 3-way stopcock, at. 84, 90 to: the, right so that the mercury 24 is withdrawn from cylinders 85 and 86. This causes the toxic agent I to be drawn into. theburette 26 through conduit 28. Next, the stopcock 84 is returned to its closed position. Subsequently, gear box I8 is set for the desired cycle, Next, chamber 42 is elevated and the animals, inserted therein, then. lowered and the suction seal be- At the end of the experiment,

should be heated by heater 3! and the walls of the spiral condenser should be heated by circulating hot. water at 3? in order to secure vaporization of the compound. From the density, the volume of liquid per minute to be displaced intothe. atomizer 219 may be calculated. The proper gear settings, determined, from the table, are made and the burette 26 is then loaded with toxic liquid I00. by withdrawing the mercury 24'. The automatic start and stop members and 51 are set ten minutes apart and the air compressorfifi is. setihto operation. The animals areplaced; in the chamber 42 and the main switch, starting the blower l5, isactuated. The clutch l1, connecting the change gear box it with gear box f2 and the motor H, is engaged causing the carriage 21.. to.

move forward, the piston 22 to move into the cylinder 23 and the toxic liquid to be displaced in the atomizer 29. The by-pass valve 38,, which is. connected to a vacuum or source of suction such. as the exhaust stack, is. open, during. this period,.so toxic vapor does-not pass into themain air stream at 4t going to the animals butbypasses chamber 42 and leaves the. system. com.- pletely, ultimately going into theexhaust system of the laboratory. However, when the carriage 2! has moved forward. sufficiently it causes. the starting member 5%} to energize the switch actuated servo-motor operated valveat 38. This causes the by-pass valve. 38- to close automatically, and the toxic material then. enters. the. main air stream at ll going to the. animals. in chamber 62. After ten minutes the stopping member 5-l-is engaged. This reopens by-pass valve 38.- and simultaneously releases the clutch l1, similarly servo-motor operated, so. that. the. delivery of toxic liquid tile is terminated. The blower I5 is continued during this period so-thatthe chamber is quickly purged. The. chamber 42 is then lifted from the glassfloor 48, the animals are. removed. Only adifierent gear setting in change gear box I8 is required to repeat the exposure just. described for a difierent time or with a difierent concentration, or different toxic material, or dif ferent mixtures of materials.

Conclusion In contrast to prior procedurewith former apparatus, the. experimental animals are inserted in. the chamber 42 of. the present invention, before the toxic material I00 enters the chamber 42 and are withdrawn after the. chamber 42- has been cleared. I

The accuracy of. the technique and. apparatus of the present invention has been tested biologicall-y and chemically with a wide. variety of compounds. Analytical samples were. taken at the vent. 45 illustrated in Fig. 1. As described herein, nominal concentrations determined either by calculation from the diameter of the piston and the distance. it moves, or bydetermination of the weight of mercurydisplaced invariably agree within 0.3%. With propionic acid, the analytical nominal ratio was of the order of 1, and with diphosgene, the analytical. nominal ratio was ofthe order of 0.95.

The advantages of the present apparatus have overcome the objections inherent in prior disclosures, as set forth herein. The saving of time by the present invention is most important, because.- at least five times: as. many compounds can be tested in aday as is possible with prior apparatus. In. the screening of compounds, where nominal concentrations are. employed, the. accuracy is greater than formerly taught because the well-known objectionable wall effects are at a minimum in an all glass apparatus. Furthermore, the material is volatilized in /5 the. total air flow inthe present apparatus,-whi1ev in former chambers not: more than /2 of the total air flow can be used. This means that substances of low volatility can be vaporized at lower tempera- E tures than in the past, and decomposition is much than the standard chamber.

less likely.

In addition to the greater speed and accuracy, the present invention. requires less material for testingand is much less hazardous to operate Furthermore, the present invention permits the dispersing of two or more toxic agents simultaneously. Since the delivery is quite independent of the vapor pressure of the substance under examination, mixtures of many materials or" various vapor pressures may easily be tested in the improved apparatus described herein.

' Theherein described illustration of the present invention shows the gear box assemblies at: [8 and at i2; the motor drive assembly at H, and the gas chamber assembly at 32, incorporated as part of the entire apparatus i0 as shown in Fig; 1 However the present invention operates very successfully as a portable unit adapted to separately engage a series of gas chambers in succession byprovidlngsuitable connections at 45 and 53 (not shown for attachment to standard connections on each chamber in a series.

Furthermore, additional standard connections can be providedat shaft M and at shaft I9'if a claims.

I claimi 1. The method of determining the toxicity of a liquid that includes the following steps: atomizing said liquid, heating said atomized liquid to form a vapor, volumetrically controlling the dilution of said vapor with air and passing said diluted vapor at a controlled rate through a sealed chamber containing animal life. 2. The method of determining the toxicity of a liquid that includes the following steps: atomizing said liquid, heating saidatomized liquid to form a vapor, volumetrically controlling the dilution of said vapor with another vapor, and passing said mixed vapors at a controlled rate through a sealed chamber containing animal life.

3. The method of determining the toxicity of a liquid that includes the following steps: atomizing said liquid, heating said atomized liquid to form a vapor, volumetrically controlling the dilution of said vapor with another vapor and with chamber, means for compressingsaid diverted 3 air, means for heating said compressed air, atomizer means in communication with said compressing means, displacement means arranged to provide a controlled metered flow of liquid to said atomizing means; selective power transmission means in operative relationship with said displacement means and with said propulsion 'means, power means for driving said transmissionmeans; vaporizing means in communication with said atomizing means, heating means in REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 843,909 Peters et al Feb. 12, 1907 1,027,823 Davis May 28, 1912 2,141,793 King Dec. 27, 1933 2,248,222 Ensign July 8, 1941 

